Versican promotes T helper 17 cytotoxic inflammation and impedes oligodendrocyte precursor cell remyelination

Remyelination failure in multiple sclerosis (MS) contributes to progression of disability. The deficient repair results from neuroinflammation and deposition of inhibitors including chondroitin sulfate proteoglycans (CSPGs). Which CSPG member is repair-inhibitory or alters local inflammation to exacerbate injury is unknown. Here, we correlate high versican-V1 expression in MS lesions with deficient premyelinating oligodendrocytes, and highlight its selective upregulation amongst CSPG members in experimental autoimmune encephalomyelitis (EAE) lesions modeling MS. In culture, purified versican-V1 inhibits oligodendrocyte precursor cells (OPCs) and promotes T helper 17 (Th17) polarization. Versican-V1-exposed Th17 cells are particularly toxic to OPCs. In NG2CreER:MAPTmGFP mice illuminating newly formed GFP+ oligodendrocytes/myelin, difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) treatment from peak EAE reduces lesional versican-V1 and Th17 frequency, while enhancing GFP+ profiles. We suggest that lesion-elevated versican-V1 directly impedes OPCs while it indirectly inhibits remyelination through elevating local Th17 cytotoxic neuroinflammation. We propose CSPG-lowering drugs as potential dual pronged repair and immunomodulatory therapeutics for MS.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Raw spatial RNA sequencing data are available at the NCBI Sequence Read Archive with the BioProject accession number: PRJNA734097 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA734097 No sample size calculation was performed. Sample size was determined based on previously published results (Ref 16,19,27), feasibility of the experiment, as well as the availability of sex and aged matched mice from transgenic mice.
A few mice in EAE experiments were excluded due to death/euthanization prior to experimental endpoint because of EAE severity.
Findings were replicated and reproduced in multiple independent experiments. The number of independent experiments and replicates used are stated in the figures of the manuscript. All experimental findings were reproducible.
All samples for experiments were randomized except for DIF treatment experiments. To have the similar mean clinical scores in both control and DIF groups, EAE mice were distributed between groups before starting the treatment according to their score .
Investigators were blinded to group allocation during EAE scoring. Blinding was not relevant for other experiments/analyses since equal experimental parameters were consistently applied for each sample during data acquisition and data analysis.
All antibodies are from commercial sources and have been validated by the manufactures. Detailed information can be found at the following links: Abcam https://www.abcam.com/primary-antibodies/how-we-validate-our-antibodies IHC and ICC determine whether an antibody recognizes the correct protein based on cellular and subcellular localization. Antibody specificity is confirmed by looking at cells that either do or do not express the target protein within the same tissue. Initially, our scientists will review the available literature to determine the best cell lines and tissues to use for validation. We then check the protein expression by IHC/ICC to see if it has the expected cellular localization . If the localization of the signal is as expected, this antibody will pass and is considered suitable for use in IHC/ICC. We use a variety of methods, including staining multi-normal human tissue microarrays (TMAs), multi-tumor human TMAs, and rat or mouse TMAs during antibody development. These high-throughput arrays allow us to check many tissues at the same time, providing uniformly as all tissues are exposed to the exact same conditions. To ensure the same, accurate results can be obtained across batches of the same antibody, we perform consistency tests to assess batch-to-batch variation. In the case of recombinant antibodies, consistency between batches is very high, meaning you are unlikely to need to perform additional optimization procedures (eg titration experiments) between batches. This may not be the case with other non-recombinant hybridoma-produced monoclonal and polyclonal antibodies where the degree of variation and drift is inherently higher. When available and suitable for assay development, recombinant monoclonal antibodies are favored and provide the best batch-to-batch consistency. We are currently working towards using KO cell lines for our ICC validation.
Amsbio https://www.amsbio.com/ultramab-antibodies/ Performance and specificity are the pre-requisites for antibodies to be used for diagnostic and therapeutic applications. AMSBIO supplies UltraMAB®, a unique line of ultraspecific and extensively validated monoclonal antibodies, to ensure superior performance in these areas. A high density protein microarray chip has been developed for antibody specificity testing, and has been applied to identify UltraMAB® antibodies.
Aveslab https://www.aveslabs.com/pages/custom-chicken-antibody-production Aves antibodies come either as an "IgY fraction" which is >90% pure antibody or as an "affinity purified antibody," which is 100% pure antibody. The IgY fraction product contains all of the antibody circulating in the hen at the time of egg production, while the affinity purified product contains only those which recognize and bind with high affinity to the antigen molecule.
BioLegend https://www.biolegend.com/en-us/reproducibility All newly developed clones at BioLegend undergo validation testing for multiple applications. This serves as a cross-check for specificity and provides clarity for research uses. Typically, antibodies are tested by two or more of the below methods. Knockout or knockdown of gene expression, such as with siRNA, is also an excellent tool for target validation. BioLegend antibodies undergo an extensive series of testing to ensure quality at every step in the manufacturing process, as well as maintaining quality after the sale.
Millipore https://www.emdmillipore.com/CA/en/life-science-research/antibodies-assays/antibodies-overview/Antibody-Development-and-Validation These efforts and collaborations have led to new validation techniques and novel antibody-based technologies, such as improved bead-based multiplex assays and imaging flow cytometry.
Novus https://www.novusbio.com/reproducibility.html Novus recognizes the need for highly validated, high quality antibodies in the life sciences community. The research community faces ongoing concerns about data reproducibility and especially the validity of antibody-based assays. A recent article in Nature discusses the variable standards and performance of antibodies and antibody suppliers in the market. Novus is committed to addressing this nature portfolio | reporting summary Note that full information on the approval of the study protocol must also be provided in the manuscript. problem and to helping our customers attain the best possible results with our products. To that end, we actively seek high quality, highly validated products and provide support to ensure that our customers have the tools to properly validate their own assays. We are also collaborating with several global initiatives that help life science researchers choose antibodies with proven results. Of the five pillars of validation established by these initiatives, genetic knockout validation provides the most reliable control for assessing antibody specificity. For technical support in validating your antibody based applications, visit technical support. R&D Systems https://www.rndsystems.com/quality/antibodies-built-for-reproducibility With the recent reports stating antibodies as one of the reasons for scientific irreproducibility, you can rest assured with our antibodies. R&D Systems® takes rigorous steps towards antibody validation and reproducibility. We have been since the beginning. For 30 years, we have used our industry-leading production standards and quality control specifications to develop antibodies that can be relied on for specificity and reproducibility. By developing and testing our products in-house, we can ensure a validated and specific antibody. We are confident in our antibodies and provide 100% guarantee for our products.
ThermoFisher/ Invitrogen /eBioscience/BD https://www.thermofisher.com/ca/en/home/life-science/antibodies/invitrogen-antibody-validation.html Part 1-Target specificity verification:This helps ensure the antibody will bind to the correct target. Our antibodies are being tested using at least 1 of the following methods to ensure proper functionality in researcher's experiments. Click on each testing method below for detailed testing strategies, workflow examples and data figure legends. Knockout-expression testing using CRISPR-Cas9 cell models Knockdown-expression testing using RNAi to knockdown gene of interest Independent antibody verification (IAV)-measurement of target expression is performed using two differentially raised antibodies recognizing the same protein target Cell treatment-detecting downstream events following cell treatment Relative expression-using naturally occurring variable expression to confirm specificity Neutralization-functional blocking of protein activity by antibody binding Peptide array-using arrays to test reactivity against known protein modifications SNAP-ChIP™-using SNAP ChIP to test reactivity against known protein modifications Immunoprecipitation-Mass Spectrometry (IP-MS)-testing using immunoprecipitation followed by mass spectrometry to identify antibody targets Part 2-Functional application validation These tests help ensure the antibody works in a particular application(s) of interest, which may include (but are not limited to): Western blotting Flow cytometry ChIP Immunofluorescence imaging Immunohistochemistry Most antibodies were developed with specific applications in mind. Testing that an antibody generates acceptable results in a specific application is the second part of confirming antibody performance.
Advanced Verification Thermo Fisher Scientific is committed to adopting validation standards for our Invitrogen antibody portfolio. The Advanced Verification badge is applied to products that have passed application and specificity testing. This badge can be found in the search results and at the top of the product specific web pages. Data supporting the Advanced Verification badges can be found in product specific data galleries.
The following stains of mice were used for this study: Female mice were used between 8 to 10 weeks of age.Female C57Bl/6J mice (6-8 weeks old) and litters from pregnant CD1 mice (P1-P2) were purchased from Charles River and used for in vitro leukocyte or OPC cultures, respectively. NG2CreER (JAX 008538 ) mice and TaumGFP (JAX 021162) mice aged 6 to 8 weeks were acquired from Jackson Laboratories and bred in University of Calgary Animal facility to produce female NG2CreER:MAPTmGFP mice (8-10 week old for EAE experiments or lysolecithin demyelination); the 2D2 TCR (TCRMOG) transgenic mice (JAX 006912) were also from Jackson Laboratories. Mice were housed between 21 and 23 degrees Celcius, in low humidity, with 12hrs. light and 12hrs. dark cycle from 7am light and starting 7pm dark.
No wild animals were used for this study.
No field-collected samples were used for this study.
All experiments were performed with ethics approval (protocol number AC21-0174) from the Animal Care Committee at the University of Calgary under regulations of the Canadian Council of Animal Care.